The cellular role of nitrogen in the biosynthesis of alkaloids by submerged culture of Claviceps purpurea (Fr.) Tul.

Asparagine was a superior nitrogen source for clavine-alkaloid production in Claviceps purpurea. Its transport into the cell excedded the cell's biosynthetic need for this amino acid. Asparagine entered the cell without degradation. This disturbed the relative pool sizes of various amino acids resulting in a change in the genetically determined ratio at which amino acids were utilized for protein synthesis. Overproduction of alkaloids (4500 mug.ml-1) may be associated with increased availability of tryptophan because of the enhanced assimilation of asparagine-derived ammonia via glutamine synthetase (EC 6.3.1.2). However, ammonium salts in the fermentation broth led to a depression of the alkaloid yield. Partial replacement of the ammonium salt by aspartic acid elevated alkaloid production.     

Origin of Epilachnapaenulata defensive alkaloids: incorporation of [1-13C]-sodium acetate and [methyl-2H3]-stearic acid

Ladybird beetles produce a large number of defensive alkaloids. Previous studies suggest that the structural diversity of these endogenous alkaloids can be traced to a common biosynthetic route based on the condensation of several acetate units. In this study, adults of Epilachna paenulata, a phytophagous neotropical species, were fed on diet enriched with potential precursors (sodium acetate, fatty acids and the amino acids lysine and ornithine) labeled with stable isotopes ((13)C, (2)H and (15)N). Labeled acetate was incorporated into the structurally related homotropane and piperidine alkaloids. The later also showed incorporation of [methyl-(2)H3] stearic acid. Our results hence support a fatty acid pathway for the biosynthesis of E. paenulata alkaloids. To our knowledge, this is the first report on the incorporation of a labeled fatty acid into a defensive piperidine alkaloid in insects.     

Effect of Tween 80 on alkaloid-producing cultures ofClaviceps paspali

Addition of Tween 80 to submerged cultures ofClaviceps paspali (Stevens and Hall) growing in a simple defined medium increased biomass formation and caused a temporary change in alkaloid synthesis intensity. The Tween-supplemented culture reached maximal alkaloid yields four days earlier than the control. The shift of alkaloid production was associated with a shift of organic acids and amino acids in the cell-pool. Thus the maximal formation of alkaloids was characterized by a decrease in the amount of citric acid, by the disappearance of succinic, fumaric and oxaloacetic acids and by increased accumulation of methionine, cysteine, alanine and histidine. The slow alkaloid synthesis was accompanied by a relatively high content of citric and succinic acids in the cell-pool and by the absence of methionine. The data are consistent with the hypothesis that ergot alkaloids participate in the regulation of the metabolism of cultures.     

Integrative Synaptic Mechanisms in the Caudal Ganglion of the Crayfish

A study of activity recorded with intracellular micropipettes was undertaken in the caudal abdominal ganglion of the crayfish in order to gain information about central fiber to fiber synaptic mechanisms. This synaptic system has well developed integrative properties. Excitatory post-synaptic potentials can be graded, and synaptic potentials from different inputs can sum to initiate spike discharge. In most impaled units, the spike discharge fails to destroy the synaptic potential, thereby allowing sustained depolarization and multiple spike discharge following single pulse stimulation to an afferent input. Some units had characteristics which suggest a graded threshold for spike generation along the post-synaptic fiber membrane. Other impaled units responded to afferent stimulation with spike discharges of two distinct amplitudes. The smaller or "abortive" spikes in such units may represent non-invading activity in branches of the post-synaptic axon. On a few occasions one afferent input was shown to inhibit the spike discharge initiated by another presynaptic input.     

Rat neurophysiological taste responses to salt solutions

1. Neurophysiological studies on the chemoresponsive tongueunits of the rat geniculate ganglion readily detected and furthercharacterized the two major functional neural groups designatedas ‘acid’ and ‘salt’ units by otherinvestigators in chorda tympani recordings. Units belongingto either of these groups were initially identified on the basisof their responses to a series of chemical solutions (a TestSeries) developed to distinguish unit groups in the geniculateganglion of the cat, dog and goat. 2. The nature of active ionicspecies effective in stimulating the units of the two differentgroups was further elaborated by studying their responses toa variety of salt solutions. From these studies, it was concludedthat the optimum molecular species for the acid units is anacid, where an acid is defined as a Br0nsted acid or protondonating molecule. A variety of molecular species may be activein solution, including some nitrogen compounds. Responses tosalt solutions by acid units are determined in part by the actionof the cation functioning as a Brønsted acid (NH₄), orin promoting proton donor molecules in water (mainly H₃O⁺).The salt units, on the other hand, are almost exclusively responsiveto solutions containing Na ⁺ and Li⁺. All other solutions werevirtually inactive. It was concluded that under a wide varietyof environmental conditions, Na+ would be the exclusive stimulusfor the salt units. Na⁺ and Li⁺ were found to be highly stimulatingwhen accompanied by a wide variety of solute anions, althoughthose containing the halogens (Cl^–, F^–, I^–,Br^–) were among the most stimulatory compounds tested.3. The establishment that the stimulus is a proton donor moleculefor the acid units and the Na⁺ and Li⁺ for the salt units hasprofound theoretical implications for the taste receptor. Biophysicalmodels of taste receptors are reviewed with respect to thisimproved understanding of the stimuli for the two types of units.It is suggested, after consideration of certain basic biochemicalaspects of the situation, that the acid receptor (possibly animidazole group) forms, as part of a protein, a proton conductingcircuit for driving an intracellular energy process devotedto proton sensing transmission. It is further suggested, inlight of the extreme specificity shown to Na ⁺, that receptorattachment is of the multidentate ligand variety and is probablylinked to Na⁺, K⁺ transport across the cell membrane, as proposedby DeSimone et al. (1981).     

Utilization of exogenously supplied 14C-saccharose into primary metabolites and alkaloid production in Catharanthus roseus

Partitioning of exogenously supplied U-¹⁴C-saccharose into primary metabolic pool as sugars, amino acids, and organic acids was analyzed and simultaneous utilization for production of alkaloid by leaf, stem, and root in twigs and rooted plants of Catharanthus roseus grown in hydroponic culture medium was determined. Twigs revealed comparable distribution of total ¹⁴C label in leaf and stem. Stems contained significantly higher ¹⁴C label in sugar fraction and in alkaloids [47 kBq kg⁻¹(DM)] than leaf. In rooted plants, label in ¹⁴C in metabolic fractions in root such as ethanol-soluble, ethanol-insoluble, and chloroform-soluble fractions and in components such as sugars, amino acids, and organic acids were significantly higher than in stems and leaves. This was related with significantly higher content of ¹⁴C in alkaloids in stems and leaves. ¹⁴C contents in sugars, amino acids, and organic acids increased from leaf to stem and roots. Roots are the major accumulators of metabolites accompanied by higher biosynthetic utilization for alkaloid accumulation.     

Expression of inwardly rectifying K+ channels in the carotid body of rat

The inwardly rectifying K+ channels, Kir1.1, Kir2.3, Kir4.1-Kir5.1, and Kir4.2-Kir5.1, are candidate chemosensory molecules for CO2/H+. Here, we determined the mRNA expression and immunohistochemical localization of these channels in the carotid body (CB) and petrosal ganglion (PG) of the rat. RT-PCR analysis revealed mRNA expression of Kir4.1 and Kir5.1 in CB, and Kir1.1, Kir4.1, and Kir5.1 in PG. Immunohistochemistry identified the glomus cells in CB to express both Kir4.1 and Kir5.1 protein, while the nerve fibers in CB were immunoreactive for Kir1.1, Kir4.1, and Kir5.1. In the PG, immunoreactivity for Kir1.1, Kir4.1, and Kir5.1 was observed in some ganglion cells. Our findings suggest that Kir channels in the peripheral chemoreceptors play a role in sensing hypercapnic acidosis and maintaining the resting membrane potentials.     

大鼠单一肌梭的电生理特征

本研究旨在观察大鼠单一肌梭的电生理特征.从大鼠比目鱼肌中分离单一肌梭,用空气隔绝法观察大鼠单一肌梭感觉末梢在不同液体环境中的放电活动.结果显示:在基础生理盐水溶液中,大鼠单一肌梭的自发放电频率很低,平均(51.78±25.63) impulse/1000s(n=13);在加有适量氨基酸的生理盐水溶液中,其自发放电频率明...     

烟草叶片对不同光质短期应答的代谢轮廓

在烟草上部叶片生理成熟期给以白、紫外A、蓝、绿、黄、红6种LED灯光处理,研究了烟草叶片对不同光质的短期应答.结果表明: 采用非靶标检测方法共检测到68种GC/MS(气相色谱/质谱)稳定的代谢产物,在PLS-DA得分图中,6种光处理的样品被明显分开;有61种在质谱库中得到鉴定,其中45种代谢物质含量在不同光处理间存在显著性差异,主要为有机酸类、糖类、TCA循环中间产物、氨基酸类等初级代谢产物;采用差异性代谢物的热力图及聚类分析将其分为5类,同时也将6个处理明显分开,其中红、蓝光处理差异最为明显,除B类物质外,其他4类均以红光处理高于蓝光处理.采用靶标性检测方法测定了3种生物碱、5种多酚及茄尼醇的含量,4种(加上非靶标测定到的烟碱)生物碱在不同光质处理间的变化趋势一致,红光和黄光明显促进了生物碱积累;莰菲醇基芸香苷和芸香苷在不同处理之间的变化趋势一致,以蓝光处理最低、黄光最高,光质对其他3种多酚的影响各异;黄光明显抑制了茄尼醇的积累,蓝光处理的叶片中茄尼醇含量最高.说明光质变化对烟草脂肪酸代谢、糖代谢、生物碱代谢、氨基酸代谢、TCA循环和莽草酸途径等多条代谢途径均有显著的影响.     

Interaction of excitatory and depressant amino acids in the frog retina

(1) Application of excitatory or depressant amino acids (concentrations from 10(-4) to 10(-2) M) could modify response patterns of the retinal ganglion cells to photic stimulus. Excitatory amino acids gave rise to spontaneous discharge while depressant amino acids inhibited spike discharge in response to test flashes. (2) Application of excitatory amino acids of more than 10(-3) M resulted in irreversible termination of spike discharges while recovery was always observed in the case of depressant amino acids even when the concentration of the applied solution was as high as 10(-2) M. No effect was observed when one exciting and one depressant amino acid were properly combined. (3) There is a mixture of four amino acids (two excitatory and two depressant) which could enhance the spike discharge in response to test flashes without giving rise to spontaneous firing. (4) It is implied that proper balance of excitatory and depressant amino acids is important in regulating the excitability of a number of neurons.     

Nitrogen metabolism in cultures of Tabernaemontana divaricata

A cell suspension culture from Tabernaemontana divaricata was fed with ¹⁵N-labelled ammonium or nitrate. The incorporation of label in free amino acids, protein amino acids and indole alkaloids was determined. Ammonium was found to be used more extensively than nitrate in the biosynthesis of these compounds. For tryptamine considerably lower labelling percentages were found than for the indole alkaloid O-acetylvallesamine and the amino acids. This indicates a vacuolar pool of tryptamine, formed at the beginning of the culture-period and not available for further alkaloid biosynthesis.     

Carbonic anhydrase enzyme histochemistry of cranial nerve primary sensory afferent neurons in the rat

Hanssons' enzyme histochemical method for the demonstration of carbonic anhydrase has been used to examine primary sensory neurons of cranial nerves in the rat (cochlear ganglion cells excluded). Numerous carbonic anhydrase positive neurons were present in the trigeminal and geniculate ganglia as well as in the mesencephalic trigeminal nucleus. A few carbonic anhydrase positive ganglion cells were found in the nodose ganglion, but none in the petrosal and vestibular ganglia. However, in the latter ganglia, satellite cells surrounding the neurons frequently showed staining for carbonic anhydrase.     

Carbonic anhydrase enzyme histochemistry of cranial nerve primary sensory afferent neurons in the rat

Summary Hansson's enzyme histochemical method for the demonstration of carbonic anhydrase has been used to examine primary sensory neurons of cranial nerves in the rat (cochlear ganglion cells excluded). Numerous carbonic anhydrase positive neurons were present in the trigeminal and geniculate ganglia as well as in the mecencephalic trigeminal nucleus. A few carbonic anhydrase positive ganglion cells were found in the nodose ganglion, but none in the petrosal and vestibular ganglia. However, in the latter ganglia, satellite cells surrounding the neurons frequently showed staining for carbonic anhydrase.     

Modulation of berberine bridge enzyme levels in transgenic root cultures of California poppy alters the accumulation of benzophenanthridine alkaloids

California poppy (Eschscholzia californica Cham.) root cultures produce a variety of benzophenanthridine alkaloids, such as sanguinarine, chelirubine and macarpine, with potent biological activity. Sense and antisense constructs of genes encoding the berberine bridge enzyme (BBE) were introduced into California poppy root cultures. Transgenic roots expressing BBE from opium poppy (Papaver somniferum L.) displayed higher levels of BBE mRNA, protein and enzyme activity, and increased accumulation of benzophenanthridine alkaloids compared to control roots transformed with a -glucuronidase gene. In contrast, roots transformed with an antisense-BBE construct from California poppy had lower levels of BBE mRNA and enzyme activity, and reduced benzophenanthridine alkaloid accumulation, relative to controls. Pathway intermediates were not detected in any transgenic root lines. Suppression of benzophenanthridine alkaloid biosynthesis using antisense-BBE also reduced the growth rate of the root cultures. Two-dimensional ¹H-NMR spectroscopy showed no difference in the abundance of carbohydrate metabolites in the various transgenic roots lines. However, transformed roots with low levels of benzophenanthridine alkaloids contained larger cellular pools of certain amino acids compared to controls. In contrast, cellular pools of several amino acids were reduced in transgenic roots with elevated benzophenanthridine alkaloid levels relative to controls. The relative abundance of tyrosine, from which benzophenanthridine alkaloids are derived, was only marginally altered in all transgenic root lines; thus, altering metabolic flux through benzophenanthridine alkaloid pathways can affect cellular pools of specific amino acids. Consideration of such interactions is important for the design of metabolic engineering strategies that target benzophenanthridine alkaloid biosynthesis.     

Quantitative changes of the alkaloid complex in a submerged culture ofClaviceps paspali

Claviceps paspali FA produced high concentrations of alkaloid under submerged conditions. Their production was found to depend on the developmental stage and treatment of the filamentous culture inoculum. A medium containing Bacto-peptone with a constant composition of amino acids was selected for the preparation of the inoculum. A two-week fermentation in a synthetic medium with mannitol at 24 ± 1 °C resulted in an increased production of total alkaloids from the original value of 100–200 μg/mL to more than 2 000 μg/mL. Addition of tryptophan did not further increase the production of alkaloids but resulted in changes of the spectrum of some metabolites. 2,3-Dihydroxybenzoic acid accompanied the alkaloids in the fermentation medium. α-Hydroxyethyllysergamide was the predominant component of extracellular alkaloids (80 % in the first days of fermentation). During fermentation the level of this alkaloid continuously decreased while the concentration of the accompanying alkaloids,i.e. lysergamide and the corresponding minor isomers, increased. An erratum to this article is available at .     

Antisense RNA-mediated suppression of benzophenanthridine alkaloid biosynthesis in transgenic cell cultures of California poppy

California poppy (Eschscholzia californica Cham.) cell cultures produce several benzophenanthridine alkaloids, such as sanguinarine, chelirubine, and macarpine, with potent pharmacological activity. Antisense constructs of genes encoding two enzymes involved in benzophenanthridine alkaloid biosynthesis, the berberine bridge enzyme (BBE) and N-methylcoclaurine 3'-hydroxylase (CYP80B1), were introduced separately into California poppy cell cultures. Transformed cell lines expressing antisense BBE or antisense CYP80B1 constructs and displaying low levels of BBE or CYP80B1 mRNAs, respectively, showed reduced accumulation of benzophenanthridine alkaloids compared with control cultures transformed with a beta-glucuronidase gene. Pathway intermediates were not detected in any of the transformed cell lines. The suppression of benzophenanthridine alkaloid biosynthesis using BBE or CYP80B1 antisense RNA constructs also reduced the growth rate of the cultures. Two-dimensional (1)H-nuclear magnetic resonance and in vivo (15)N-nuclear magnetic resonance spectroscopy showed no difference in the abundance of carbohydrate metabolites in the various transgenic cell lines. However, transformed cells with reduced benzophenanthridine alkaloid levels contained larger cellular pools of several amino acids including alanine, leucine, phenylalanine, threonine, and valine compared with controls. The relative abundance of tyrosine, from which benzophenanthridine alkaloids are derived, was less than 2-fold higher in antisense-suppressed cells relative to controls. These results show that alterations in the metabolic flux through benzophenanthridine alkaloid biosynthesis can affect the regulation of amino acid pools. These data provide new insight into the metabolic engineering of benzophenanthridine alkaloid pathways.     

The primary structure of the core protein of the small, leucine-rich proteoglycan (PG I) from bovine articular cartilage

Two forms of small, interstitial proteoglycans have been isolated from bovine articular cartilage and have different core proteins, based on NH2-terminal analysis and peptide mapping (Choi, H. U., Johnson, T. L., Pal, S., Tang, L-H., Rosenberg, L. C., and Neame, P. J. (1989) J. Biol. Chem. 264, 2876-2884). These proteoglycans have been called PG I and PG II. Since they were first described, they have also been called "biglycan" (PG I), "decorin," and "DS-PG" (PG II). This report describes the primary structure of PG I from bovine articular cartilage. The protein core consists of 331 amino acids with a molecular mass of 37,280 Da. The amino acid sequence shows 55% identity to the cDNA-derived sequence of PG II from bovine bone. There are four discrete domains in the amino acid sequence. Domain 1, at the NH2 terminus (approximately 23 amino acids), contains two sites of attachment of dermatan sulfate, both of which match the consensus sequence of Asp/Glu-X-X-Ser-Gly-hydrophobic. Neither of these sites is substituted to 100% with glycosaminoglycan in native PG I. Domain 2, near the NH2 terminus and containing approximately 28 amino acids, has a cysteine pattern similar to a domain near the COOH terminus of mouse metallothionein and contains at least one disulfide bond (between the first and fourth cysteine residues). The majority of the core protein of PG I (domain 3) is a leucine-rich domain containing ten repeating units (approximately 231 amino acids). Patthy [1987) J. Mol. Biol. 198, 567-577) has shown that for PG II, the majority of domain 3 shows considerable similarity to leucine-rich alpha 2-glycoprotein (LRG) from serum. Domain 2 of PG I or PG II also has an analog in LRG, in that it has two cysteines in a similar place. The major motif in the PG I described here, in PG II and in LRG, is a series of leucine-rich repeats. PG I and PG II both contain 10 leucine-rich repeats which are 14 amino acids long and which are somewhat irregularly spaced, while LRG contains 9 leucine-rich repeats spaced 10 amino acids apart. Other proteins which contain leucine repeats are the platelet glycoprotein Ib, which is involved in platelet adherence to subendothelium (eight repeats in the alpha chain and two in the beta chain), the protein encoded by the Toll gene (involved in lateral and ventral spatial organization in Drosophila) and chaoptin (a protein involved in Drosophila photoreceptor morphogenesis).(ABSTRACT TRUNCATED AT 400 WORDS)     

Stress-related polyketide metabolism of Dioncophyllaceae and Ancistrocladaceae

The discovery of a novel biosynthetic pathway to isoquinoline alkaloids is described. The naphthylisoquinoline alkaloid dioncophylline A, one of the most prominent representatives of a new class of structurally and pharmacologically intriguing secondary metabolites, is shown to originate from acetate units, both molecular halves, the isoquinoline part and the naphthalene portion, being formed from identical polyketide precursors. All other tetrahydroisoquinoline alkaloids previously investigated, ultimately originate from aromatic amino acids. The novel pathway to isoquinoline alkaloids (hence acetogenic) was proved by feeding experiments with (13)C-labelled precursors administered to callus cultures of Triphyophyllum peltatum (Dioncophyllaceae), followed by NMR investigations using the potent cryoprobe methodology. The new pathway is largely stress-sensitive: upon exposure to chemical, biotic or physical stress, T. peltatum stops producing the isoquinoline part, so that the naphthalene moiety accumulates in the chemical form of naphthoquinones like plumbagin and droserone and the chiral tetralone isoshinanolone.     

Effects of bicarbonate buffer on acetylcholine-, adenosine 5'triphosphate-, and cyanide-induced responses in the cat petrosal ganglion in vitro

Acetylcholine (ACh), adenosine 5'-triphosphate (ATP) and sodium cyanide (NaCN) activate petrosal ganglion (PG) neurons in vitro, and evoke ventilatory reflexes in situ, which are abolished after bilateral chemosensory denervation. Because in our previous experiments we superfused the isolated PG with solutions free of CO2/HCO3- buffer, we studied its effects on the PG responses evoked in vitro. PGs from adult cats were superfused at a constant pH, with HEPES-supplemented (5 mM) saline with or without CO2/HCO3- (5%/26.2 mM) buffer, and carotid (sinus) nerve frequency discharge (fCN) recorded. Increases in fCN evoked by ACh, ATP and NaCN in CO2- free saline were significantly reduced (P < 0.05, Wilcoxon test) when CO2/HCO3- was present in the superfusion medium. Thus, the presence of CO2/HCO3- buffer appears to reduce PG neurons sensitivity to ACh, ATP and NaCN, an effect that may underlie the lack of ventilatory reflexes after bilateral chemodenervation.     

A highly sensitive taste receptor cell for pyrrolizidine alkaloids in the lateral galeal sensillum of a polyphagous caterpillar, Estigmene acraea

Adult males of Estigmene acraea use pyrrolizidine alkaloids to produce pheromones and all stages probably use pyrrolizidine alkaloids for defense. The alkaloids are obtained from plants by the caterpillars. We demonstrate that a contact chemoreceptor neuron in the lateral galeal sensillum exhibits a dose-dependent response to seneciphylline N-oxide, a widely occurring pyrrolizidine alkaloid, down to concentrations of 10(-9) x mol l(-1), and even at 10(-12) x mol l(-1) the response is greater than to salt alone. At concentrations of 10(-6) mol x l(-1) and above the instantaneous firing rate is very high, and at 10(-4) mol x l(-1) initially exceeds 500 spikes s(-1). The firing rate declines in the 200 ms following stimulus onset but then is sustained with an instantaneous firing rate in excess of 100 spikes s(-1) for at least the next 800 ms. At lower concentrations a delay occurs before firing is initiated, and then the pattern of firing is irregular. The cell is equally sensitive to some but not all of several other pyrrolizidine alkaloids tested as free bases and their N-oxides. It also responds to ouabain, which may also serve as a defensive compound, and to asparagine and fructose but with much higher thresholds than to the pyrrolizidine alkaloids.